Staphylococcus aureus, (MRSA), Escherichia coli, and Pseudomonas aeruginosa. This study investigated (i) the PPIX-MED-mediated antimicrobial photodynamic effect on these three species in vitro and (ii) the effect of antimicrobial photodynamic therapy (aPDT) combined with the use of an antibiotic on the healing in vivo of third-degree burns of rats with the wounds infected by these bacterial species. PPIX-MED exerted a potent inhibitory effect on the growth of the three bacterial species by producing reactive oxygen species when photoactivated. PPIX-MED-mediated antimicrobial photodynamic therapy (PPIX-MED-aPDT) had high bacterial photoinactivation ability in vitro, with a minimum inhibitory concentration of 15.6 μM PPIX-MED against each of the three types of bacteria and minimum bactericidal concentrations of 31.25 μM against MRSA and E. coli and 62.5 μM against P. aeruginosa. In rats with third-degree burns infected by a mixture of these bacteria, the bactericidal efficiency of PPIX-MED–aPDT-combined-with-antibiotic treatment was higher than that of antibiotic or aPDT treatment alone. This was confirmed by analysis of viable bacterial counts in wound tissue and blood. Enzyme-linked immunosorbent assay revealed that aPDT-combined-with-antibiotic treatment resulted in an obvious reduction in tumor necrosis factor-alpha and interleukin-6 levels compared with the no-treatment control group and the other treatment groups. Immunohistochemistry revealed that the expression of basic fibroblast growth factor and CD31 (a marker of neovascularization), expressed in burn wound tissue was higher in the aPDT-combined-with-antibiotic treatment group than in the other groups. PPIX-MED–aPDT has a promising bactericidal effect both in vitro and in vivo, and PPIX-MED–aPDT-combined-with-antibiotic treatment enhanced the healing of infected third-degree burns in rats.Cationic porphyrin conjugate, protoporphyrin IX-methyl ethylenediamine derivative (PPIX-MED) has a potent photosensitive antibacterial effect on clinically isolated bacteria, including methicillin-resistant  Staphylococcus aureus (MRSA), Escherichia coli, and Pseudomonas aeruginosa are early colonizers, accounting for the majority of burn wound infections  . aPDT inThe photosensitizer is the crucial element in aPDT. Cationic photosensitizers are capable of efficiently killing Gram-negative bacteria, inhibiting the secretion of inflammatory factors, and promoting wound healing after infection of burns . We hypoin vivo on healing of experimental rat burn wounds infected with a mixture of pathogenic bacteria. The wounds were treated by aPDT using the photosensitizer PPIX-MED .A semiconductor 650-nm laser  was selected for this study, and the light spot energy density was determined with an optical power meter .–5 M dye in 96-well microtiter plates were sealed with cover slips to avoid evaporation. Photobleaching measurements at 300–800 nm were conducted for 30 min at ambient temperature, and data were recorded every 5 min. The delivered light energy was 0.2 J/cm2 per min, and the total light energy density was 6 J/cm2.Photobleaching was conducted with a Multimode Microplate Spectrophotometer . BrieflyΔ]) experiments were performed according to Δ] was determined by the decomposition of 1,3-diphenyl isobenzofuran (DPBF) in DMSO; [ΦΔ] correlated with the decay of the absorption of DPBF at 410 nm  used as the reference (Singlet oxygen quantum yield ( was injected under the eschar of the wound of rats in groups B and D. Then, after 30 min, these rats were illuminated with a 650-nm laser for 10 min . On the next day, the same light dose was given to increase the effect of PPIX-MED–aPDT, but no more PPIX-MED was administered. This treatment is defined as one treatment. The above-mentioned treatment was repeated four times . In groups C and D, the rats underwent a tail intravenous injection of ceftriaxone 0.8 ml once every 2 days; the above-mentioned treatment was repeated four times.At 24 h after infection, 100 μl of PPIX-MED solution  for PPIX-MED reached 0.69 when a mixture of DPBF (2 × 10–5 mol/l) and PPIX-MED was irradiated with a 650-nm semiconductor laser (6 J/cm2). The DPBF decay rate was higher in the presence of PPIX-MED than TPP, revealing that PPIX-MED was more efficient than TPP in producing singlet oxygen.An ideal photosensitizer should achieve a high singlet oxygen yield. In this study, DPBF was used as a reducing agent to trap singlet oxygen  were incubated with PPIX-MED in the dark for 30 min at 37°C and then exposed to light . The concentration of PPIX-MED required to make the suspensions change visibly from turbid to clear was regarded as the MIC, while the concentration at which five colonies or less were observed on plates was regarded as the MBC. As shown in E. coli, and P. aeruginosa. The MBC values were 31.25 μM for MRSA, 31.25 μM for E. coli, and 62.5 μM for P. aeruginosa. Meanwhile, the dark toxicity of PPIX-MED was relatively low, with MIC and MBC values > 500 μM for the three strains. From the MBC values, it can be concluded that PPIX-MED–aPDT is very effective against E. coli, P. aeruginosa, and MRSA. Thus, PPIX-MED is a good compound to evaluate in the treatment of bacterially infected burn wounds.The MIC and the MBC of PPIX-MED toward the test bacteria were studied. Bacterial suspensions , the photoinactivation effect of PPIX-MED against MRSA, E. coli, and P. aeruginosa was PPIX-MED dose dependent. A sharp decrease in bacterial survival fraction was observed with an increase of the PPIX-MED concentration. The activity of the bacteria was largely inhibited in the concentration range 0–31.25 μM PPIX-MED, and the inhibition rate was >99% at 31.25 μM for E. coli and P. aeruginosa (which are Gram-negative bacteria) and 15.6 μM for MRSA (a Gram-positive bacterium). The results reveal that PPIX-MED displayed highly efficient photoinactivation toward these three bacterial species.To delve into the photoinactivation effects of PPIX-MED against MRSA, E. coli, and P. aeruginosa. The phototoxicity of PPIX-MED was concentration dependent (2), the NIH 3T3 cell activity exceeded 10.6% at 31.25 μM PPIX-MED, at which concentration the three bacterial strains were almost fully eliminated.NIH 3T3 fibroblast cells were used as an example of normal mammalian cells to assess the phototoxicity of PPIX-MED via the MTT assay. NIH 3T3 cells were incubated with PPIX-MED in identical conditions to those used for MRSA, ependent . In darkE. coli, P. aeruginosa, or MRSA incubated with PPIX-MED was consistent with the dose-dependent photoinactivation effects.Uptake of PPIX-MED by the three species of bacteria was examined by confocal laser scanning microscopy. Porphyrin conjugates emit red fluorescence at 650 nm when excited at 405 nm, which can be readily monitored with a fluorescence microscope system . The imaTo monitor the wound healing process, the leakage quantity of burn wounds, the presence of secretions, and healing range were observed at different times after infection. At 1 day after infection, the wounds of rats in the treated groups and the control group (no treatment) showed pale necrosis, the skin became hard and convex, and the wound surface showed bloody scabs and necrotic tissue coverage and congestion. At 4 days after infection, the untreated (control group) animals showed wound decay and ulceration. However, in the aPDT group and the aPDT-combined-with-antibiotic group, the skin was smooth, the wound scab was black and dry, and the wounds were better than in the antibiotic-only treatment group. At 10 days after infection, the wounds of the rats in the untreated control group were beginning to heal, but they were still very large and there was pus under the scab. Wound healing in the antibiotic-only treatment group was better than that in the untreated group. The scab did not fall off. It was gray, and there was pus underneath. In the aPDT and aPDT-combined-with-antibiotic treatment groups, all the scabs fell off, and new pink tissues were exposed.in vivo aPDT experiments, about five or 10 times the MIC is generally chosen as the working dose . The rats in the aPDT + antibiotic therapy group exhibited faster wound healing than those in the antibiotic-therapy-only group (p < 0.01) or the aPDT treatment group. On day 17 post-infection, the wounds in the aPDT + antibiotics group had healed (96.7 ± 1.5); the healing rate was significantly better than that in the antibiotic-only treatment group (p < 0.01) and the aPDT treatment group, respectively. The healing rate of the aPDT treatment group was superior to that of the antibiotic-only treatment group (p < 0.01). Therefore, aPDT combined with antibiotics was most efficient in the treatment of mixed bacterial infection of burn wounds, and aPDT treatment was also somewhat effective.For ing dose . On the P. aeruginosa, and E. coli at each observation time point; on days 4, 10, and 14 post-infection, all the treatment groups exhibited an obvious reduction in bacterial viability compared with the controls (p < 0.05). In the treatment groups, bacterial viability decreased in the order aPDT + ceftriaxone group > aPDT group > ceftriaxone group. This suggests that PPIX-MED–aPDT has a bactericidal effect against MRSA, P. aeruginosa, and E. coli in burn wounds, and aPDT combined with antibiotics is more efficient in the treatment of burn infection.The viability of bacteria in wound tissue was determined as an index of the bactericidal effect of PPIX-MED–aPDT treatment . The conp = 0.004). At 14 days, the rate of bacteria-positive blood cultures was still zero for the aPDT + ceftriaxone group and was 11.1% for the aPDT group and 42.8% for the ceftriaxone group; however, the rate in the control group remained high (80%).As shown in In the control group, five rats died on days 1–4 post-infection. In the antibiotic treatment group, four rats died on days 1–3. In the aPDT treatment group, one rat died (on day 1). All rats in the aPDT + ceftriaxone group survived the experiment. These data indicate that PPIX-MED–aPDT was the most efficient approach to the treatment of mixed bacterial infection in a burn wound .p < 0.01) compared with the no-treatment (control) group and the other treatment groups; at a given time point, the level of TNF-α and IL-6 in the groups was control > antibiotic > aPDT > aPDT + antibiotics. These results suggest that aPDT, especially when combined with antibiotic therapy, can inhibit the expression of IL-6 and TNF-α in burn tissues, which will reduce the inflammatory reaction in the tissues and help with wound healing.ELISA was used to investigate tissue TNF-α and IL-6 concentrations in skin tissue after treatment . The TNFp < 0.05). At 10 days, the values were 41.33 ± 3.50, 91.00 ± 7.27, and 99.67 ± 9.27, respectively, compared with 16.67 ± 2.66 for the control group (p < 0.05). At day 17, the expression of bFGF in the antibiotic-only (48.33 ± 17.95), aPDT-only (51.83 ± 14.95), and aPDT + antibiotics (43.67 ± 4.97) groups remained higher than that in the control group (39.00 ± 5.90).p < 0.01). The expression of bFGF in the aPDT treatment group was higher than that in the antibiotic treatment group . The expression level in the control and the antibiotic groups increased throughout the healing process. However, in the aPDT treatment group and the aPDT + antibiotic group, bFGF expression first increased and then decreased, being highest at 10 days post-infection among the timepoints that we measured: 99.7 ± 9.3 in the aPDT + antibiotic treatment group and 91.0 ± 7.3 in the aPDT group.At 4 and 10 days post-infection, the bFGF expression level in the aPDT + antibiotic group was obviously higher than that in the antibiotic and the aPDT treatment groups (p < 0.01) (p < 0.01). The MVD in the aPDT-only treatment group was significantly higher than that in the antibiotics-only group (p < 0.01 at 4 and 10 days post-infection). The new MVD in the aPDT + antibiotic group reached its observed maximum on day 10.On days 4, 10, and 17 post-infection, the new blood vessel density (MVD), determined by the expression of CD31, was significantly higher in the treatment groups than in the control group ( < 0.01) . MVD in in vivo toxicity of PPIX-MED–aPDT and aPDT + antibiotics treatments using the organ index in rats (p < 0.01), which may be caused by the toxicity of ceftriaxone sodium to the liver.We examined the potential  in rats . We harvIn control group rats, the body weight was lower than it was in the treatment groups. When dissecting the rats, many infections were observed on the spleen, and the small intestine adhered to the abdominal wall on both sides. Rats in the antibiotic-only treatment group also showed symptoms such as a grossly enlarged spleen and intestinal adhesions. These symptoms indicated that the experimental animals had different degrees of bacterial organ infections.Complications caused by drug-resistant bacterial infection after burns are a major factor in the death of patients. Bacterial attachment to an open wound during the healing process is a prominent etiological factor of sepsis and multiple organ dysfunction syndrome (MODS), for example . As suchIn vitro  group or the group treated with antibiotic alone. Moreover, half the rats in the control (untreated) group died within 4 days of infection, and 40% of the rats in the antibiotic-only treatment group died early in the experiment (days 1–3), but no rats in the aPDT + antibiotics treatment group died, and only one died in the aPDT-only group (on the first day after burning and infection). Immunohistochemical studies showed that aPDT and aPDT combined with antibiotic promoted a high expression of bFGF before day 10; on day 10, the expression of bFGF was lower, whereas the expression level in the control group and the antibiotic-only group continued to increase at that timepoint. This may suggest that aPDT and aPDT combined with antibiotic can promote fibroblast proliferation that enhances wound healing .The viable bacteria in wound tissue and in blood were determined as an index of the bactericidal effects of aPDT and aPDT combined with antibiotics. On the 4th, 10th, and 14th day after infection, the aPDT-combined-with-antibiotics treatment group exhibited an obvious reduction in bacteria compared with the control group and the aPDT-only treatment group and the antibiotic-only group. This experiment confirmed that aPDT can significantly reduce the number of bacterial colonies under phase callus, increasing the rate of wound healing, and promote healing efficacy.p < 0.01). In the aPDT-combined-with-antibiotics group, the new blood vessel density reached its observed maximum on day 10 and was lower on day 14. The results showed that aPDT-combined-with-antibiotic treatment can promote neovascularization in burn wounds. TNF-α is a crucial contributing factor to inflammation-mediated pathophysiology . The pleThe major advantages of aPDT are its specific effect on target cells, a lack of collateral effects, activity only on exposure to light, and inability of bacteria to develop resistance to this type of killing . FurtheraPDT and aPDT-combined-with-antibiotic treatment showed beneficial effects in accelerating the healing process of bacterially infected third-degree burns of rats. These treatments stimulate macrophages to release chemical mediators, cytokines, and growth factors, which, in turn, stimulate and increase the production of connective tissue and create a new supply of blood vessels to nourish the wound site and promote remodeling. There was a histological tendency for better cicatrization after the use of aPDT combined with antibiotics in burn healing. The synergistic effect of aPDT-combined-with-antibiotic treatment could be promising for the management of third-degree burn skin infections.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by the Laboratory Animal Management Committee/Laboratory Animal Welfare Ethics Committee, Institute of Radiation Medicine, Chinese Academy of Medical Sciences.TL and GH performed conceptualization. ZZ, TL, and GH performed data curation. ZZ and GH performed formal analysis. GH performed funding acquisition, project administration, and supervision. JZ, ZZ, and GH performed investigation. LZ, TL, and GH performed methodology. ZZ, TL, and GH performed resources. ZX, YW, JM, and GH performed validation. ZZ and GH performed visualization and writing – original draft. ZZ, TL, and GH performed writing – review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


COVID-19 is an emerging respiratory disease caused by a novel coronavirus. There is not much evidence assessing the knowledge of dental surgeons regarding COVID-19. This study was conducted to assess the knowledge of COVID-19 among dental surgeons of Nepal.P-value < 0.05 was considered statistically significant.A web-based cross-sectional study was conducted among registered dental surgeons of Nepal. Ethical approval was obtained. Consent was taken, and knowledge on COVID-19 was assessed via a pre-tested structured questionnaire using Google form. The form was emailed to the participants. Descriptive analysis was performed using frequency, percentage, median and inter-quartile range. Man-Whitney test and Kruskal-Wallis tests were carried out to see the difference in knowledge score. p = 0.013); M.D.S.  graduate and B.D.S. graduate (0.041); dental surgeons of government healthcare facilities and other healthcare facilities (p <  0.001); dental surgeons of COVID-19 centers and non-COVID-19 centers (0.002).Total 227 dental surgeons responded to the questionnaire . Almost two-third ( 65.2% ) of the respondents were B.D.S.  graduates. Only 29.1% worked in the government hospitals. 17.6% were currently involved in COVID-19 management. Of the participants, 87.7% knew about the condition of the requirement of Personal Protective Equipment (PPE) but only 29.1% could correctly answer the framed question for PPE. The median knowledge score calculated was 14.0 (8.0–18.0). The bivariate analysis showed a statistically significant difference in knowledge score among the age group ≥30 years and < 30 years (The dental surgeons of Nepal have a good knowledge of COVID-19, and they can be utilized for assisting in the management of COVID-19 cases in Nepal.The online version contains supplementary material available at 10.1186/s12879-020-05620-4. Coronavirus disease COVID-19) is an emerging respiratory disease caused by a novel coronavirus 9 is an e. WHO decCOVID-19 is largely spread by human-to-human transmission through droplet infection with some cases of feco-oral and direct transmission . The incThe doctor-patient ratio of Nepal is 0·17 per 1000 population—substantially less than the WHO recommendation of 2·3 doctors per 1000 population .The numbA web-based quantitative descriptive cross-sectional study was carried out from 10 March to 25 March, 2020. This study considered (95% CI) and 95% power to estimate the sample size. For this purpose, 65.7% (p) was considered from a study  conducteA pre-tested structured questionnaire using Google form was emailed to collect data from registered dental surgeons of Nepal. Ethical approval was taken from the Ethical Review Board (ERB) of Nepal Health Research Council (Reg. no.: 2383). The Google forms emailed had informed consent form with details of the study. A reminder email was sent to all the previously emailed dental surgeons after a week of the first email. Confidentiality of the information was ensured by maintaining privacy.Age of the participants was categorized as less than 30 and more than or equal to 30. The gender of the participants was categorized as male and female. The degree of the dental surgeons was categorized as Bachelor of Dental Surgery (B.D.S.) and Master of Dental Surgery (M.D.S.). The working province of the participants included all the seven provinces of Nepal in which Karnali and Sudurpaschim Province were merged for bivariate analysis. Type of hospital/ health center where the dental surgeons worked was categorized as Government, Private clinic, Private hospital, and semi-government. The level of hospital/health center was categorized as Primary, Secondary and Tertiary levels. Working hospitals of the dental surgeons was categorized into COVID-19 center and non-COVID-19 center. Involvement in COVID-19 management was categorized into “Yes” and “No”. Received training on COVID-19 management was categorized into “Yes” and “No”.For assessment of knowledge on COVID-19 among dental surgeons of Nepal, a 14-items questionnaire tool was developed from the published literatures –12. FaceP-value < 0.05 was considered statistically significant.All the responses from Google forms were exported to Microsoft Excel 2016 and were exported to SPSS version 23  software. Descriptive analysis was performed using frequency, percentage, median and inter-quartile range. After the normality test of the knowledge score, it was found that the overall knowledge score was non-normal (skewed). Man-Whitney test (for variables with two categories) and Kruskal-Wallis test (for variables with more than two categories) were carried out to see the difference in knowledge score between different socio-demographic variables. This was a web-based survey conducted to assess the knowledge of COVID-19 among dental surgeons of Nepal. Total 227 dental surgeons responded to the questionnaire . Almost two-third (65.2%) of the respondents were B.D.S. graduates. More than 80% of the respondents were from three provinces . It was seen that only 29.1% of the dental surgeons worked in government hospitals, and more than 50% of the dental surgeons worked in a tertiary level health care facility. More than one-third (35.7%) of the dental surgeons are currently working in a health facility that is a COVID-19 center. It was seen that 17.6% of dental surgeons are currently involved in COVID-19 management. Only 28.2% of dental surgeons received COVID-19 management training, but the majority of them had received online training (71.8%) , but only 29.1% could correctly answer the framed question for PPE. Similarly, 93.8% knew about the preventive measures (quarantine and isolation), but only 59.5% could correctly answer the framed questionnaire .p = 0.013). The knowledge score of male dental surgeons was more compared to female dental surgeons, but it was not statistically significant (0.063). M.D.S. trained dental surgeons had more knowledge of COVID-19 compared to B.D.S. trained dental surgeons, which was statistically significant (0.041). The dental surgeons working in government healthcare facilities had better knowledge scores compared to other healthcare facilities, and it was statistically significant (p <  0.001). Similarly, the dental surgeons working in COVID-19 centers had higher knowledge compared to non-COVID-19 centers, and the difference was statistically significant (0.002). The dental surgeons involved in the management of COVID-19 patients had better knowledge scores than the ones not involved in COVID-19 management, and it was statistically significant (0.004). But, there was no statistically significant difference between the knowledge score of dental surgeons who received training on COVID-19 and who did not (p = 0.853)  indicates a good knowledge of COVID-19 among dental surgeons of Nepal which was similar to the study conducted by Kamte et al.  The majo.0 indicaThis was a nation-wide study and it was promising to know that almost all the dentists were aware of the origin of COVID-19. The high infectivity of COVID-19 has alarmed to keep the healthcare personals safe. If not so, then the infected asymptomatic healthcare personals might be the carrier and be the medium of transmission. Hence, PPE has been recommended by WHO to be used compulsorily while dealing with suspected/diagnosed cases of COVID-19 .p = 0.013). During the postgraduate training, the residents are trained about research and are updated with knowledge based on recent evidences. In contrast, very few institutions are seen to engage their undergraduates in such learning practices [p = 0.041). These findings were similar to those of Kamte et al. [p < 0.001). It was a similar situation among Jordanian dentists where 22.8% were working in public service whereas it was only 6.82% of north Italian dentist associated with National health System [In the medical field, an increase in age and experience improves the knowledge of the clinicians. The senior clinicians are mostly postgraduates. They have better knowledge about research, and their practices are mostly evidence-based , 20. It ractices . This mie et al.  and Gupte et al. . The denh System , 17. Simp = 0.853).Traditionally seminar, workshops, hands-on training with face to face interactive sessions was used for regular updates on recent evidence. The outbreak of COVID-19 has limited all the mass gathering and social events to decrease the transmission rates. The recent evidence suggests that online training can be of similar significance compared to on-site training, but further exploration is needed to establish it as fact , 24. It The strength of the study is that, it was a nation-wide study where the participating dental surgeons were from all the provinces. The calculated sample size was obtained, and the results can be generalized throughout Nepal. This study had some limitations, the web-based cross-sectional nature of the study design that is prone to selection bias and information bias on the respondent’s side.In conclusion, the dental surgeons of Nepal have a good knowledge of COVID-19. Based on the finding of this study, dental surgeons can be utilized for assisting in the management of COVID-19 cases when needed. But, they are to be oriented and trained before, about management protocols of COVID-19.Additional file 1. Questionnaire.


However, the volumetric capacitance is low, which hinders its practical application. Secondly, we incorporate activated carbon (AC) into HGNS-900 to make a composite EDLC material. The effect of the AC:HGNS-900 ratio on the capacitance, high-rate performance, and cycling stability are systematically investigated. With a proper amount of HGNS-900, both the electrode gravimetric and volumetric capacitances at high rate charging/discharging are clearly higher than those of plain AC electrodes. The AC/HGNS-900 composite is a promising electrode material for nonaqueous EDLC applications.We demonstrate a facile and effective method, which is low-cost and easy to scale up, to fabricate holey graphene nanosheets (HGNSs) via ultrafast heating during synthesis. Various heating temperatures are used to modify the material properties of HGNSs. First, we use HGNSs as the electrode active materials for electric double-layer capacitors (EDLCs). A synthesis temperature of 900 °C seems to be optimal, i.e., the conductivity and adhesion of HGNSs reach a compromise. The gravimetric capacitance of this HGNS sample (namely HGNS-900) is 56 F·g With the increasing demand for energy storage devices in modern society, supercapacitors (SCs), including electric double-layer capacitors (EDLCs) and pseudocapacitors, represent an attractive energy storage technology owing to their higher power density, wider operating temperature window, superior cycling stability, and greater charge–discharge efficiency compared to secondary batteries ,2. EDLCsAmong various nanostructured carbons, GNSs are very promising because of their high surface area, great electrical conductivity, excellent mechanical stability, and stable chemical/electrochemical properties ,14. A vaHGNSs can not only be the main electrode active materials, but can also act as an electrode additive in EDLCs. Some earlier reports have suggested that combinations of various carbon materials with different structural features and physicochemical properties can give rise to better EDLC performance compared to that of their single-component counterparts due to some synergistic effects ,26,27. HIn the present work, we present a facile and effective method, which is low-cost and easy to scale up, to fabricate HGNSs. The effects of the heating temperature on the HGNS material properties are investigated. Applications of HGNSs for the main electrode active material and for mixing with AC particles to modify the electrode performance are both studied. The AC/HGNS composite is found to be a good potential electrode material for nonaqueous EDLCs. With the incorporation of the proper amount of HGNS, both the gravimetric and volumetric capacitances at high charge–discharge rates are significantly higher than those of plain AC electrodes. 2 gas was released at a high rate that built up high pressure between the neighboring graphitic layers. This not only exfoliated the graphite but also punched holes at structurally weak points on the graphene. As a result, holey graphene nanosheets (denoted as HGNS-300) were obtained. These HGNSs were then heated under an argon/hydrogen (5:1) mixed atmosphere at a rate of 60 °C·min−1 to 700 °C, 900 °C, 1100 °C, respectively, and held for 1 h. The samples are denoted as HGNS-700, HGNS-900, and HGNS-1100, respectively. The AC powder was supplied by China Steel Chemical Corporation  and was used as received. HGNSs were synthesized using a modified Staudenmaier method . Briefly2 adsorption/desorption isotherms were measured at 77 K. The specific surface area was calculated using the Brunauer–Emmett–Teller (BET) method.The microstructures were examined using scanning electron microscopy  and transmission electron microscopy . To study the crystal structures of various samples, X-ray diffractometry  was performed. A Raman spectrometer  was used to analyze the carbon bonding characteristics. X-ray photoelectron spectroscopy  was employed to probe the surface chemical compositions of the samples. The NN-methyl-2-pyrrolidone solution. The slurry was pasted onto etched Al foil and vacuum-dried at 120 °C for 8 h. The obtained electrode was roll-pressed and punched to match the required dimensions of a CR2032 coin cell. Two symmetrical electrodes divided by a NKK cellulose separator were assembled into a coin cell. The electrolyte was composed of 1 M tetraethylammonium tetrafluoroborate  salt in propylene carbonate  solvent. The coin cell assembly was conducted in an argon-filled glovebox (Innovation Technology Co. Ltd.), where both the moisture and oxygen content levels were maintained at below 0.3 ppm. Cyclic voltammetry (CV) and galvanostatic charge–discharge tests were performed using a Solartron 1470E potentiostat within a cell voltage range of 0–2.5 V. For each condition, at least five coin cells were measured. The capacitance deviation was typically less than 5%. The reported data are the median values. Electrochemical impedance spectroscopy (EIS) was used to characterize the cell internal resistance. The frequency range and potential amplitude, regulated with a BioLogic VSP-300 potentiostat, were 105–10−2 Hz and 10 mV, respectively. The electrode slurry was prepared by mixing 80 wt % active material, 10 wt % carbon black, and 10 wt % poly(vinylidenedifluoride) in The TEM images in The crystallinity of various samples was characterized by XRD. In −1, respectively. Raman spectroscopy was employed to investigate the carbon bonding structures of various HGNS samples. The in-plane vibration of the graphitic lattice correlates to the G band, whereas the disorders such as impurity, imperfection, and sp2 domain boundaries are associated with the D band . The D aAs shown in XPS was also used to examine the surface chemistry of the HGNSs. The survey scans in −1. It can be observed that the HGNS-900 and HGNS-1100 electrodes exhibited more rectangular CV curves, indicating better capacitive behavior compared to the HGNS-300 and HGNS-700 electrodes. It is believed that the removal of the oxygen-containing functional groups on HGNSs can reduce the electrode internal resistance, promoting electrochemical performance. −1. The HGNS-300, HGNS-700, HGNS-900, and HGNS-1100 electrodes exhibited specific capacitances of 17, 25, 56, and 53 F·g−1, respectively. A temperature of 900 °C appeared to achieve capacitance optimization. It was reported that the oxygen-containing functional groups can contribute some pseudocapacitance [In order to evaluate the electrochemical properties of various HGNS electrodes, symmetrical cells with a TEABF4/PC electrolyte were assembled. acitance ,35. We sinter.) between the HGNSs [inter. values of various cells. The HGNS-300 and HGNS-700 cells exhibited Rinter. values of 100 and 75 Ω, respectively. The high resistance is responsible for the unsatisfactory electrochemical properties shown in inter., which is in line with the oxygen concentration variation trend shown in inter. values for the HGNS-900 and HGNS-1100 cells were 26 and 16 Ω, respectively.he HGNSs . Table S−3) than those of the other electrodes, as shown in It was observed that the active material coating layer of the HGNS-1100 electrode was not as uniform as the others, and its adhesion with the Al current collector was relatively poor. While casting the electrode slurry with a doctor blade gap of 50 µm, the final thickness of the HGNS-1100 layer after drying was ~9 µm, which is considerably lower than that (~30 µm) of the other electrodes. This is probably related to the fact that the oxygen-containing functional groups had been removed to a greater degree. Thus, the bonding force between the HGNSs became weaker. Moreover, the HGNS-1100 film density was lower (~0.140 g·cm−1) of GNSs were reported for thin electrodes [−1 at 30 µm thickness. One reason for the low capacitance is the restacking nature of graphene sheets for a thick electrode [−3). Accordingly, the HGNS-900 alone is not an ideal electrode material for EDLCs.In the literature, high gravimetric capacitances , high density, and low cost; however, its electronic conductivity is not sufficient. We speculated that the incorporation of HGNS-900 may modify the properties of the AC electrode. In this section, AC was mixed with HGNS 900 in various proportions . The surface morphologies of the electrodes were examined by SEM; the obtained low-magnification images are shown in However, mixing HGNS-900 with AC is worthy of consideration. AC has high specific surface area . This reflects the fact that the AC particles agglomerated after slurry coating and drying. Additionally, the electrode binder may block the pores of AC, thereby reducing the surface area. The surface area for AC/HGNS-900 (20:1) reached 968 m2 ·g−1, indicating that the incorporation of HGNS could alleviate the AC aggregation and pore blocking problems. However, with further increasing the HGNS-900 ratio, the BET surface area decreased ) because the intrinsic specific surface area of HGNS-900 is lower than that of AC. The surface area of AC/HGNS-900 (5:1) was reduced to 635 m2·g−1, since the AC surface was covered by agglomerated graphene sheets that shielded the AC pores  are shown in −1), the optimal AC:HGNS-900 ratio seems to be 20:1, because the electrode surface area and conductivity have been improved (vs. the plain AC electrode) at the same time. The AC:HGNS-900 ratio of 10:1 is more favorable for higher rate applications (>10 A·g−1), revealing that the incorporation of HGNS can effectively promote the electrode charge–discharge kinetics. A proper amount of meso- and macro-pores  and AC/HGNS-900 (10:1) cells showed Rinter values of 15 and 10 Ω, respectively. The HGNSs act as the conducting pathways that surround and connect the AC particles, reducing the electrode resistance. This explains the improved high-rate performance of these electrodes. However, when the AC:HGNS-900 ratio became 5:1, the Rinter. increased to 20 Ω. This can be attributed to the discontinuity of the electrode layer, as shown in −1. As shown, the capacitance retention ratios for all electrodes were 91–95% after 10,000 cycles. The Incorporation of HGNS did not significantly affect the electrode cycling stability. The durability of supercapacitors is a crucial factor for practical applications. −1) and the film density is inadequate (0.156 g·cm–3) at a practical electrode thickness of 30 µm. It was concluded that HGNS-900 alone is not a competitive electrode active material. However, HGNS-900 can be an effective performance promoter for EDLC AC electrodes. The AC:HGNS-900 ratio is a crucial factor affecting the distribution of HGNSs within the electrode, and thus, the resulting charge–discharge performance. If the HGNS-900 is excessive, the HGNSs agglomerated, shielded the AC pores, and separated the AC particles, deteriorating the electrode performance. However, a proper amount of HGNS-900 can alleviate the AC aggregation and create conducting pathways within the electrode. As a result, both the gravimetric and volumetric capacitances at high charge–discharge rates are considerably higher than those of the plain AC electrode. The appealing performance of the AC/HGNS-900 composite has shown great potential for high-performance energy storage applications.A cost-effective method exploiting ultrafast heating during GO reduction was employed to produce HGNSs. It was found that the heating temperature played a role in determining the internal resistance, capacitance, and processability of the HGNS electrodes. However, even for the optimal HGNS-900 electrode, the specific capacitance is still unsatisfactory (56 F·g


DI SPECT allows for an automatic and accurate estimation of absorbed doses, introducing true personalized dosimetry. However, photon crosstalk between isotopes can compromise image quality. This study investigates the effect of 99mTc downscatter on 166Ho dosimetry, by comparing 166Ho-SPECT reconstructions of patient scans acquired before (166Ho-only) and after additional administration of 99mTc-colloid (166Ho-DI).Accurate dosimetry is essential in radioembolization. To this purpose, an automatic protocol for healthy liver dosimetry based on dual isotope (DI) SPECT imaging, combining holmium-166 , was calculated based on the administered therapeutic activity.The 166Ho-only or 166Ho-DI SPECT (kappa = 0.093). Quantitative analysis indicated that the mean absorbed dose difference between 166Ho-DI and 166Ho-only was − 2.00 ± 2.84 Gy , − 5.27 ± 8.99 Gy , and 0.80 ± 1.08 Gy  for VOIHEALTHY, VOITUMOR, and VOILUNGS, respectively. The corresponding Pearson’s correlation coefficient between 166Ho-only and 166Ho-DI for absorbed dose was 0.97, 0.99, and 0.82, respectively.The qualitative assessment showed no distinct clinical preference for either The DI protocol enables automatic dosimetry with undiminished image quality and accuracy.Clinicaltrials.gov (NCT02067988) on 20 February 2014.The clinical study mentioned is registered with  Radioembolization requires a comprehensive initial safety evaluation  and assessment of intrahepatic microspheres distribution for dosimetric evaluation. Pre-treatment image-based dosimetry enables radioembolization optimization, because it allows assessment of the biodistribution of microspheres in the liver, which is often heterogeneous and clustered. Because absorbed dose and treatment outcome (toxicity and efficacy) are correlated, dosimetry should ultimately lead to improved patient selection and individualized treatment planning , was chosen as the metric for comparison since it was deemed the most clinically relevant parameter. The dose difference between 166Ho-only SPECT/CT images were considered as reference standard. Inter-observers’ agreement was measured by means of Cohen’s kappa statistic (κ) , were reported. LoA are computed as mean ± coefficient of reproducibility (CRP), equal to 1.96 × standard deviation. The linear correlation between absorbed dose in 166Ho-only and 166Ho-DI was expressed in terms of the Pearson’s correlation coefficient r. In addition, two-sided paired t test (at α = 0.05) was performed to check the statistical difference between mean absorbed dose based on 166Ho-only and on 166Ho-DI SPECT .In each comparative analysis, the stic (κ) , for the166Ho-SPECT reconstructions were considered reliable for a diagnostic purpose. Based on their preference for either 166Ho-only or 166Ho-DI, their inter observer agreement (Cohen’s kappa coefficient) was equal to 0.09 . The Cohen’s κ value, close to 0, shows the lack of agreement on a favorite imaging option (166Ho-only or 166Ho-DI). Moreover, both were considered suitable for diagnostic use. Therefore, no distinct preference for either one of the reconstructions for use in clinical practice could be concluded.According to the qualitative assessment carried out by two expert nuclear medicine physicians, all TUMOR and of 7 procedures for the VOIHEALTHY, since they did not satisfy this requirement. The lower limit for the VOI volume was introduced to ensure an adequate dose recovery. The included dataset is reported in Table The constraint in volume for the VOIs ≥ 25 ml) led to the exclusion of 21 procedures for the VOI5 ml led The results, for each of the three VOIs, for both the dataset considered entirely and split among pre- and post-treatment data, are reported in Table HEALTHY, VOITUMOR, and VOILUNGS are shown in Fig. 166Ho-DI and 166Ho-only was negative for the VOIs within the liver, while it was slightly positive for VOILUNGS. CRP was equal to 5.56 Gy for VOIHEALTHY, 17.62 Gy for VOITUMOR, and 2.12 Gy for VOILUNGS.Bland-Altman plots for VOI166Ho-only and 166Ho-DI, assessed using Pearson’s correlation coefficient , 0.97 for VOIHEALTHY (r = 0.96 for pre- and r = 0.98 for post-treatment data), and 0.82 for VOILUNGS (r = 0.72 for pre- and r = 0.89 for post-treatment data).The linear correlation between T test p value results are < 0.00001, 0.00035, and < 0.00001 for VOIHEALTHY, VOITUMOR, and VOILUNGS, respectively.166Ho reconstruction derived from a DI acquisition was investigated. The inter-observer agreement (κ = 0.093) indicated no specific preference for either the 166Ho-only or DI acquisition in the qualitative analysis. The quantitative analysis demonstrated a good correlation between 166Ho-only and 166Ho-DI with a Pearson’s correlation coefficient > 0.95 for both VOIHEALTHY and VOITUMOR and 0.82 for VOILUNGS. The difference between mean absorbed dose between 166Ho-only and 166Ho-DI SPECT was statistically significant for all VOIs ; however, the mean difference was considered clinically not relevant. The limits of agreement for the difference between 166Ho-DI and 166Ho-only were deemed acceptable by experienced nuclear medicine physicians. Because assessments of dose to the tumor, healthy liver, and lungs serve a different purpose clinically, physicians defined different acceptable limits of agreement for each category prior to this study. A mean difference of 2 Gy with a limit of agreement of ± 5 Gy was considered adequate for healthy liver assessment (median absorbed dose for 166Ho-only VOIHEALTHY: 27 ± 7.08 Gy), being the dose-limiting factor for radioembolization treatments. A less restrictive value may be applied for the absorbed dose in the tumor because the clinical range for efficacy is variable and not well defined (median absorbed dose for 166Ho-only VOITUMOR:: 116 ± 44.91 Gy). With respect to the lungs, according to 166Ho-miscrosphere instructions for use [166Ho-only and 166Ho-DI was defined at approximately 3 Gy. This overestimation prevents underestimation of the lung absorbed dose (median absorbed dose for 166Ho-only VOILUNGS: 3 ± 0.99 Gy). For all VOIs, 95% of the data was well within the corresponding clinically acceptable limits of agreement.In this study, the qualitative and quantitative accuracy of a 166Ho-DI and on 166Ho-only in the VOILUNGS, the lungs were delineated on the corresponding attenuation correction LDCT. However, the lungs were not always entirely visible within the field of view of the SPECT. This drawback is negligible in case the lung perfusion is homogeneous, but this assumption is not always correct [LUNGS were more affected by this drawback. This explains the lower Pearson’s correlation coefficient (0.82) and the higher number of outliers.Even though rarely encountered (< 1%) , radiati correct , 20. In The use of the proposed DI protocol has potential benefits, amongst which the possibility to (semi) automatically identify and delineate tumor and healthy tissue within a single SPECT/CT acquisition. Additionally, simultaneous acquisition of both isotopes avoids the registration difficulties, both being time consuming and prone to additional errors in dosimetry.99mTc distribution or 166Ho accumulation can be processed to obtain an automatic delineation of the regions of interest. Healthy liver might be delineated on the 99mTc reconstruction while tumor lesions presenting focused 166Ho uptake can be delineated on the 166Ho image. The definition of these two compartments is a requirement for the use of the partition model [SPECT images showing either on model . This me99mTc images. Because of the need to apply the same VOIs to both 166Ho-only and 166Ho-DI for tumor and healthy liver, a co-registration process was involved. This can lead to small misalignments, which may have impacted on the mean absorbed dose difference. In addition, tumors with a volume smaller than the 25 ml, not considered for this study, could be more affected by registration related error and partial volume effect which will further hamper the tumor dosimetry quantification.Some limitations apply to this study. All images were acquired with the same SPECT/CT scanner, which restricts the used k-factor to this imaging setup. Nonetheless, it is possible to extend this study to other scanners . Furthermore, the liver VOIs were manually segmented to ensure an adequate volume definition, similar among all datasets (as can be seen in Table 99mTc-colloid. To compensate for the lungs region just partially covered by the SPECT field of view, it could be possible to implement a two-bed position protocol to cover both the lungs and the abdominal region. An assessment of 99mTc crosstalk impact on 166Ho acquisitions stratified by volume of interest dimensions could provide a better insight in the possibility to perform 166Ho-DI acquisition without being hampered by an increase effect of partial volume effect due to the presence of an additional isotope.To cope with the mentioned limitations, some future steps can be taken. A phantom experiment could help to determine the accuracy with which it is possible to recover the absorbed dose at different activity concentration, both with and without the presence of 166Ho-only SPECT/CT acquisition has a beneficial effect on patients, decreasing the discomfort related to an imaging procedure that takes half an hour. Further analysis is required to implement a clinical workflow to automatically process information derived from the DI protocol and obtain personalized planning for radioembolization.The possibility to skip the 166Ho-DI SPECT can be safely used instead of a 166Ho-only acquisition. The differences between the 166Ho-only and 166Ho-DI protocol reconstructions were considered to be clinically acceptable, and thus the dual isotope protocol can be adopted in clinical practice.Based on a qualitative, as well as a quantitative analysis on patient data, a Additional file 1. Supplementary materials.


LPIN1-related acute recurrent rhabdomyolysis (RM), first reported in 2008, is an autosomal recessive inherited metabolic disease. In recent years, LPIN1 gene variants have been identified as one of the main causes of severe RM in children in Western countries. The disease is extremely rare in China, and we report a case of acute recurrent RM caused by a novel compound heterozygous LPIN1 variant.LPIN1 gene of the proband: the paternal c.2047A > G(p.I683V) was an unreported missense variant, and the maternal c.2107_2108 insAGG(p.Q703delin sQE) was an unreported in-frame variant.A 15-year-old Chinese boy presented with myalgia after strenuous exercise, accompanied by transient increases in serum creatine kinase and myoglobin and persistent hyperuricaemia and hyperbilirubinaemia. Genetic analysis using high-throughput genomic sequencing and Sanger sequencing revealed that there was a compound heterozygous variant in the LPIN1 variants should always be considered in the differential diagnosis. The clinical features of our case are atypical, which highlights the importance of an accurate diagnosis by genetic testing. If detected early, the condition may be controlled, and the prognosis may be improved.In children with RM,  LPIN1 gene encoding LPIN1 are the second most common cause of early childhood recurrence and severe RM, second only to mitochondrial fatty acid ß-oxidation defects (FAOs). LPIN1 is an intracellular protein that controls metabolism by acting at multiple regulatory levels. This soluble protein acts at the endoplasmic reticulum to dephosphorylate phosphatidic acid (PA) to form diacylglycerol (DAG), penultimate steps in Kennedy pathway of triacylglycerol (TAG) synthesis. LPIN1 also acts in the nucleus to directly interact with DNA-bound transcription factors to regulate gene expression [LPIN1-related acute recurrent RM was first reported by Zeharia et al. in 2008. It is an autosomal recessive inherited metabolic disease that is most commonly triggered by fever, exercise, fasting, and anaesthesia. Severe RM can cause acute kidney failure and may lead to death [LPIN1 whose gene variant points are different from previous cases.Rhabdomyolysis (RM) is a clinical entity characterized by the destruction of skeletal muscle with the resultant release of intracellular content into the bloodstream that leads to systemic complications . The clapression . LPIN1-rto death . To the 2 (reference: 17.1–23 kg/m2), and muscular tone and power in both lower extremities were normal. We suspected this was as characteristic of RM. Then, the boy was advised to take oral sodium bicarbonate to alkalize urine, fructose and vitamin C to improve metabolism and promote cell repair until another health examination 1 month later. We found that the boy’s levels of CK, myoglobin, bilirubin and uric acid CK were higher than the previous levels. The results of these serum biochemical test are also shown in Table A 14-year-old boy went to the hospital for a health examination, which detected a slight increase in bilirubin that was followed up regularly. Then, the boy suffered from an intermittent fever, mainly a moderate-to-high fever, lasting for 1 week. After a health examination, we found that the boy’s levels of CK, myoglobin, bilirubin and uric acid were all significantly increased. Serum biochemical test results are shown in Table LPIN1 gene variants in this proband included the missense variant c.2047A > G(p.I683V) and in-frame variant c.2107_2108 insAGG(p.Q703delin sQE). The c.2047A > G variant of the 2047th nucleotide in the coding region from adenine to guanine results in the 683rd amino acid changing from isoleucine to valine; c.2107_2108 insAGG changed by way of indels, and the 703rd amino acid changed from glutamine (Q) to glutamine (Q) and glutamate (E), which caused the protein length to change  of 233 genes related to neuromuscular diseases  for urine alkalization when myalgia and dark urine occur. Under our guidance, his daily life and studies have not been affected.Although the result of genetic testing was uncertain regarding pathogenicity, we communicated the possibility of LPIN1-related acute recurrent RM is a life-threatening disease that is characterized by the age of the first onset being younger than 6 years old and the peak CK level at the onset exceeding 10,000 IU/L [LPIN1 gene (p.I683V and p.Q703delin sQE) in a Chinese boy with acute recurrent RM, and his clinical characteristics are quite different from those reported in previous studies. Our proband is a 15-year-old adolescent with a later age of onset and only one episode of mild RM. His serum CK and myoglobin were normal in between episodes, but there was long-term hyperuricaemia and hyperbilirubinaemia. To the best of our knowledge, we report the third case of LPIN1-related acute recurrent RM in a patient of Chinese ethnicity, while the case with hyperuricaemia and hyperbilirubinaemia is shared for the first time (MIM#268200).000 IU/L . We discLPIN1” as keywords. There were 57 patients (including our case) with complete clinical data who were genetically diagnosed with acute recurrent RM caused by the LPIN1 gene variant [LPIN1-related acute recurrent RM was more common in males than in females (28/57 49.1% vs. 24/57 42.1%). The first onset was before the age of 6 for the most part, and the number of episodes was usually 3 or fewer. The peak CK level was often greater than 10*104U/L. In 40 cases, potential trigger factors had been reported, among which fever was the most common (26/40 65.0%)          enzymes that catalyse a key reaction in glycerolipid biosynthesis [LPIN1 gene is located on chromosome 2p25.1 with two highly conserved domains: the N-terminal LPIN (N-LIP) region spans the first 108 residues, and the C-terminal LPIN (C-LIP) domain consists of 624–830 amino acids. The C-LIP domain contains two key domains: the PAP catalytic motif Asp-Xaa-Asp-Xaa-Thr (DXDXT (689–693)) and the transcriptional coactivator motif Leu-Xaa-Xaa-Ile-Leu (LXXIL (678–682)) [LPIN1-related RM.The LPIN protein family is composed of three members, LPIN1, LPIN2, and LPIN3, encoded by the ynthesis . Only LPynthesis . The LPI78–682)) , 26. The78–682)) –19, 22. 78–682))  found th78–682)) . HoweverLPIN1 has transcriptional coregulatory activity, which, through association with peroxisome proliferator-activated receptor alpha (PPARα) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), regulates lipid metabolism and the mitochondrial respiratory chain (mRC) . DespiteLPIN1 gene variants showed a significant decrease in LPIN1 levels and PAP activity, but the adipose tissue appeared to develop normally and had normal lipid reserves. The levels of metabolic components such as triglycerides and cholesterol remained unremarkable in the serum in our case, consistent with the results of previous studies. Our patient underwent muscle-related examinations, such as electromyography and calf muscle MRI, and the results were not significant, consistent with the report of Minton et al. [LPIN1-associated RM. Moreover, it should be noted that our case is easily confused with phosphoglycerate kinase (PGK) deficiency, which is a rare cause of congenital hemolytic anemia accompanied by hyperbilirubinaemia, muscle weakness, myalgia and myoglobinuria [Pelosi et al.  found thn et al. , which sn et al. . We specn et al. . High sen et al. . Becauseobinuria . AlthougLPIN1-related RM were advised to use high-concentration glucose solutions early to provide calories, which reduced the duration of RM from the typical 7–10 days to 5 days. Meijer et al. [LPIN1 variants. However, whether high-concentration glucose solutions and dexamethasone can effectively treat RM remains to be further explored in the future.Our patient has been taking supportive treatments at home, such as rest, rehydration, energy supplementation, a low-purine diet, and supplements to alkalize urine, and he is living well at present. Symptomatic treatment of RM with LPIN1 deficiency mainly includes early detection, active hydration, high-energy carbohydrate intake, and monitoring of hyperkalaemia and arrhythmia . To avoir et al.  successfLPIN1-related acute recurrent RM is more common in males. The first onset is before the age of 6, and the number of episodes is usually within 3. The peak CK level is often greater than 10*104U/L. Fever, infection and fasting are the most common trigger events, and gene variants are common as homozygous variants, while the Chinese cases being reported all involve compound heterozygous variants. As the mortality rate due to LPIN1 deficiency is as high as 14.0% and some patients have atypical symptoms, as in our case, it highlights that paediatric clinicians in the Departments of Cardiology, Neurology, Nephrology, and Immunology should promptly detect the disease and treat it symptomatically. Genetic testing, a method to clearly diagnose and analyse the source of pathogenic genes, is increasingly recognized. Our research is of reference value for an in-depth understanding of the clinical and laboratory characteristics of LPIN1-related acute recurrent RM and for improving the early diagnosis and clinical management of patients with this disease.In summary, we diagnosed a Chinese boy with acute recurrent RM and discovered the novel compound heterozygous variants c.2047A > G(p.I683V) and c.2107_2108 insAGG (p.Q703delin sQE). According to a systematic literature review,


Early life stress can exert detrimental or beneficial effects on neural development and postnatal behavior depending on the timing, duration, strength, and ability to control the stressors. In this study, we utilized a maternal and social deprivation (MSD) model to investigate the effects of early life stress on neural stem cells (NSCs) and neurogenesis in the adult brain. We found that MSD during the stress-hyporesponsive period (SHRP) (early-MSD), when corticosterone secretion is suppressed, increased the size of the NSC population, whereas the same stress beyond the SHRP abrogated these effects. Early-MSD enhanced neurogenesis not only in the dentate gyrus of the hippocampus, one of the classic neurogenic regions, but also in the amygdala. In addition, mice exposed to early-MSD exhibited a reduction in amygdala/hippocampus-dependent fear memory. These results suggest that animals exposed to early life stress during the SHRP have reinforced stress resilience to cope with perceived stressors to maintain a normal homeostatic state. Environmental manipulation during postnatal life predisposes the mammalian brain to altered function and cellular remodeling resulting in behavioral and mood changes. One of the cellular mechanisms contributing to these alterations is adult neurogenesis, which occurs in two neurogenic regions, the olfactory bulb and the dentate gyrus of the hippocampus, in the normal rodent brain . Neural The size of the neural precursor population and the extent of adult neurogenesis are under the influence of many external stimuli . For exaMaternal separation (MS) is a well-characterized rodent model for early life stress but shows significant dissimilarities in its effects between studies; this could be due to differences in the start or end of its timing, duration of the separation episodes, and strength of the stressors . For insThe Animal Care and Use Committee of the Shiga University of Medical Science, in compliance with the guidelines of the National Institutes of Health, approved the use and care of animals in this study. Pregnant C57BL/6J mice were provided on gestational day16 from Japan SLC, Inc.  and their pups with total number of 224 were used in this study. We used only male mice for the behavioral studies and both male and female mice for other experiments. All animals were maintained at room temperature on a 12:12 h light–dark cycle (8 am–8 pm).The day pups were delivered was defined as postnatal day (P) 0. The pups were randomly assigned to one of the following experimental groups: (a) separation from the dam and littermates from P1 to P14 during the SHRP (early-MSD); (b) separation from P15–P21 after the SHRP (late-MSD); and (c) without separation. For the neurosphere and corticosterone assay experiment, an extended-MSD (P1–P21) group was added. MSD and littermate control pups were bred in the same dams. The MSD pups were separated from their dam and littermates for 3 h (1–4 pm). They were placed into paper cups with 8 cm bottom-diameter and 20 cm height, lined with wood-chip bedding. To avoid disturbing the dam by the vocalization of the MSD pups, the cages containing the pups’ cups were moved to a different room from their home cage. The non-separated control mice were undisturbed from their dam except for routine cage changes. On P28, all pups were weaned and group-housed with littermates of the same sex.2, 2 mM CaCl2, 26 mM NaHCO3, 0.18% glucose, and 100 unit/ml penicillin/streptomycin) and the entire SEZ of the lateral ventricles was excised and cut into small pieces. The tissue was subjected to enzyme digestion in a solution containing 1.33 mg/ml trypsin, 0.67 mg/ml hyaluronidase, and 0.2 mg/ml kynurenic acid  for 1 h at 37°C to facilitate dissociation of the tissue. The tissue was then triturated using a fire-polished Pasteur pipette in serum-free media  containing trypsin inhibitor (Roche). After washing with SFM, the cells were cultured at a density of 5 cells/μl in a 24-well plate  in 500 μl of SFM containing 20 ng/ml EGF, 10 ng/ml FGF2, and 2 μg/ml heparin . The number of floating spherical colonies (neurospheres) was counted after 6 days when the spheres possessed a diameter >0.08 mm. Total numbers of neurosphere-forming NSCs in the adult brain were calculated from the following: volume in which SEZ cells were suspended , volume applied in the culture, and number of resultant neurospheres in the culture.The isolation of neural stem cells (NSCs) from adult brains was performed as previously described . Brieflyg for 15 min. The corticosterone levels were assayed by ELISA  according to the manufacturer’s protocol with slight modification at a dilution of 1:1000.Blood was collected at the end of separation on P14 for the early-MSD group and on P21 for the early-, late-, and extended-MSD groups. The blood was allowed to clot undisturbed for 1 h and was centrifuged at 2,000 × Immediately after the last MSD at P14, the mice were injected with a single dose of 5-ethynyl-2′-deoxyuridine (EdU)  and sacrificed after 2 h. After deep anesthesia with 60 mg/kg ketamine and 12 mg/kg xylazine in PBS, the mice were transcardially perfused with 4% paraformaldehyde solution. Their brains were removed and post-fixed with the same fixative overnight, cryoprotected with 20% sucrose in PBS at 4°C, and then cryosectioned coronally at a thickness of 20 μm using a Leica cryostat (CM3050S). The sections were permeabilized with 0.3% Triton X-100 for 5 min followed by Click-iT EdU labeling using Alexa Fluor-555 azide  for 30 min at room temperature according to the manufacturer’s protocol. Then, after extensive washing with PBS containing 0.05% Tween-20 (PBS-T), the sections were incubated with Hoechst 33258  for 30 min.®-conjugated secondary antibodies  at a dilution of 1:4000 overnight at 4°C, followed by Hoechst 33258  for 30 min.P15 mice were injected with 5-bromo-2′-deoxyuridine (BrdU)  every 3 h for a total of five injections and sacrificed 6 weeks after the last injection. After deep anesthesia, the mice were transcardially perfused with 4% paraformaldehyde solution. Brains were removed and post-fixed with the same fixative overnight, cryoprotected with 20% sucrose in PBS at 4°C, and then cryosectioned coronally at a thickness of 20 μm using a Leica cryostat (CM3050S). The sections were irradiated in a microwave for 5 min in 10 mM citrate buffer (pH 6.0) and heated to over 90°C, followed by permeabilization with 0.3% Triton-X-100 for 5 min and then blocking with 10% normal goat serum in PBS-T for 1 h. The primary anti-BrdU rat monoclonal antibody , used at a 1:200 dilution alone, with an anti-NeuN mouse monoclonal antibody  at a 1:500 dilution or with anti-GFAP mouse monoclonal antibody at a 1:1000  and anti-S100β rabbit polyclonal antibody at 1:500 , was applied to sections for overnight incubation at 4°C. Then, after extensive washing with PBS-T, the sections were incubated with Alexa Fluor+ cells in the SEZ and SGZ or BrdU+ cells in the SEZ, DG, and BLA was determined using the optical dissector method  test was conducted according to manufacturer’s protocol . The OF apparatus is a 40 cm-square gray arena enclosed by 30 cm-high walls which are housed in a soundproof chamber and the center of the floor is illuminated at 70 lux. Ten-week-old male mice were individually placed in the center of the arena and allowed to explore freely for 10 min. Their behavior was recorded using a video tracking system and infrared photobeam sensors. The horizontal activity , vertical activity (number of rearing behaviors measured by counting the number of photobeam interruptions), and time spent in the central region comprising 36% of the total area were analyzed using tracking software . The total distance was used as an index of locomotor activity in a novel environment, rearing numbers were used to assess stereotypic behavior, and the percentage of time spent in the central area was used as an index of anxiety.The contextual and cued fear conditioning was measured using an O’Hara & Co., Ltd., Tokyo, Japan, apparatus. Conditioned training and testing sessions were conducted during the daytime in a soundproof behavioral room. Conditioned fear memory was evaluated by contextual and cued tests. The contextual test was used as a hippocampus-dependent learning task, while the cued test was used as a hippocampus-independent learning task. The chamber used for the conditioning training and contextual test was a rectangular clear Plexiglas chamber . The chamber floor consisted of 46 stainless steel rods with a diameter of 2 mm, placed 4 mm apart. A shock generator was connected to the rods via a cable harness. The cued test chamber was a white opaque triangular chamber . Both chambers were placed in a soundproof box facing a ceiling-mounted CCD camera connected to a video monitor. The training (conditioning) session consisted of three tone-shock pairings. Sixteen-week-old mice were placed into the bright rectangular chamber and allowed to explore freely for 2 min. A tone stimulus  was presented as the conditioned stimulus (CS) for 30 s, followed by a mild foot shock  as the unconditioned stimulus (US). Two more CS–US pairings were given at 2-min intervals. The contextual and cued fear conditioning test was conducted at 1 and 21 days after the training to assess recent and remote fear memory, respectively. In the contextual test, the mice were replaced into the same context as the conditioning chamber for 5 min without receiving the foot shock and auditory cue. One hour after the context testing, the cued test was conducted in an altered context. Mice were placed in a dark triangular chamber for 5 min  and then the auditory cue was turned on for 3 min (cued phase). One day after the remote memory testing, extinction training was performed. Mice were exposed to the same procedure as in the training session but without shocks. The extinction training was repeated on three subsequent days. One day after the last extinction training, the contextual/cued test was employed to assess extinction. Freezing behavior was defined as a lack of movement for >2 s, which was measured by video tracking software . The percentage of time spent freezing was used as an index of fear memory during all sessions.Spatial memory was assessed using the Barnes maze test  according to the manufacturer’s protocol. On a white circular platform with a diameter of 1 m, 16 holes were equally distributed around the perimeter, one of which was equipped with an escape box. The Barnes maze test was begun at 16 week old in place of the fear conditioning test. After habituation to the apparatus, each mouse was placed at the center and, over a period of 5 min, the time and number of errors required to enter the escape box was measured. Three trials per day were conducted for eight consecutive days. On day 9, a 3-min probe trial was conducted without the escape box to confirm that the spatial memory of the target was acquired based on navigation by distal environmental room cues. As for the reversal task, the target escape box was moved to a new position opposite the original one. After the training trials for another eight consecutive days, the mice received a probe trial.t-test or analysis of variance (ANOVA). Statistical analysis was performed using GraphPad Prism 6 software for Macintosh . Comparisons between the data from two groups were analyzed using an unpaired t-test. Multiple group comparisons were made with one-way or repeated-measured analysis of variance (ANOVA) followed by post hoc Tukey’s test when significant main effects were detected. The normal distribution of the data was checked by Shapiro–Wilk test and if the normality failed, non-parametric Mann–Whitney U test or Kruskal–Wallis test followed by post hoc Dunn’s multiple comparisons test was used. All data are presented as the mean ± SEM and a p-value of <0.05 was considered statistically significant.Data were analyzed using Student’s F = 20.49, p < 0.0001; time effect, F = 315.2, p < 0.0001, post hoc Tukey’s multiple comparisons test). However, the early-MSD pups caught up with the control pups until 6 weeks old . Then, we sought to determine whether or not the size of the NSC population in the adult brain was altered by early life stress, which may show dissimilar effects depending on differences in the timing and duration of the separation episodes. At 6 weeks of age, we performed a colony-forming neurosphere assay in a low cell-density culture condition, in which single NSCs clonally generate neurospheres  = 6.818, p = 0.0011).We first isolated each pup from its dam and littermates between P1–P14 (early-MSD), P15–P21 (late-MSD), or P1–P21 (extended-MSD) and measured bodyweight of the pups at P7, P14, and P21 to evaluate the effect of MSD on their growth rate. While the bodyweight was comparable among groups until P14, it was significantly higher in the late-MSD (11.71 ± 0.22 g) and significantly lower in the early- (7.34 ± 0.28 g) and extended-MSD (5.70 ± 0.50 g) compared to control group (10.04 ± 0.43 g) (ospheres . We fount(10) = 0.9476, p = 0.3657), the levels after the last stress significantly increased in the late- and extended-MSD mice compared to controls  = 7.483, p = 0.0017; post hoc Tukey’s multiple comparisons test: control vs. late-MSD, p = 0.0086; control vs. extended-MSD, p = 0.0096), suggesting that high corticosterone levels elicited by stress after P14 in late- and extended-MSD abrogated the increase of NSC population size observed in early-MSD. On the other hand, early MSD showed comparable levels of the corticosterone to controls at P21 , suggesting that the stress during the SHRP did not activate the HPA-axis and, therefore, that early- and late-MSD impose different long-term impacts on the neural development. We further investigated whether or not the effects of early-MSD on NSC population size persist long into adulthood by means of the neurosphere assay. We found that the total number of NSCs in the SEZ remained higher at 21 weeks in mice from the early-MSD group than the control group, after the completion of behavioral tasks described below (t(6) = 2.504, p = 0.0463).We speculated that the HPA axis response to the stress was activated in late- and extended-MSD mice after P14. Indeed, while the corticosterone levels were comparable at P14 between the early-MSD and control mice (+ cells were comparable between early-MSD and control groups (t(8) = 0.5056, p = 0.6286 for SEZ; t(8) = 0.7786, p = 0.4586 for SGZ). Most of the labeled cells are considered vigorously dividing transit amplifying cells (TACs), progeny of NSCs, because NSCs become relatively quiescent and have much longer cell cycle times than TACs. Therefore, it is possible that the expansion of NSC population was not reflected to the number of EdU+ cells at this point. Additionally, no EdU+ cells were detected in other brain regions, including the BLA in both groups.Next, we examined numbers of proliferating cells immediately after early-MSD by injecting EdU and labeling dividing cells for 2 h . We foun+ cells that remain in the SEZ at 8 weeks largely consist of NSCs. Results showed that early-MSD significantly increased the number of BrdU+ cells in the whole SEZ compared to controls (t(22) = 2.483, p = 0.0211). These results were further confirmed by triple immunostaining for BrdU, GFAP, and S100β because NSCs are GFAP+ S100β– whereas astrocytes are GFAP+ S100β+ (+ GFAP+ S100β– cells in the early-MSD mice compared to controls (t(9) = 5.755, p = 0.0003). Neural precursor cells are also present in the SGZ of the dentate gyrus, which migrate radially to the granular cell layer and differentiate to express neuronal markers. Quiescent NSCs that have incorporated BrdU at P15 should remain in the SGZ and retain the label at 8-week-old and they can be identified as GFAP+ S100β– radial glia-like cells (+ GFAP+ S100β– cells in the SGZ was significantly higher in the early-MSD than the control (t(9) = 3.065, p = 0.0135). Thus, early life stress during the SHRP increases the size of the NSC population both in the SEZ and SGZ.The NSC population was assessed by long-term BrdU incorporation: BrdU was administered on P15 and BrdU-positive cells in the SEZ were assessed at 8 weeks of age. We used BrdU, instead of EdU, because BrdU seems less cytotoxic than EdU for long-term stem cell labeling . During + S100β+ . We obseke cells . We reve+ cells in the whole dentate gyrus (t(9) = 7.950, p < 0.0001) and more BrdU+ NeuN+ cells in the granular cell layer (t(9) = 8.851, p < 0.0001) were observed in the hippocampus of mice after early-MSD than control. Because EdU incorporation into the SGZ cells immediately after the last separation of early-MSD was comparable to the control (+ NeuN+ cells were observed after early-MSD (t(9) = 4.680, p = 0.0012). Since no EdU+ cells were detected in the amygdala at P14, those BrdU+ NeuN+ cells may have incorporated BrdU somewhere outside the amygdala and migrate to it. However, our data do not exclude a possibility that our 2-h labeling was not sufficient to label the neurogenesis at P14 mouse amygdala. Indeed, in situ neurogenesis in the adult amygdala has been reported  = 4.137, p = 0.0193; post hoc Tukey’s test: early-MSD vs. late-MSD, p = 0.0170). However, neither group showed significant differences compared to controls; therefore, we concluded that the growth retardation, if any, would not significantly affect behavioral analyses. First, we conducted an OF test, in which we evaluated the total traveled distance, the number of rearing behaviors and time spent in the center area  = 0.7626, p = 0.0009; post hoc Tukey’s test: control vs. late-MSD, p = 0.0020; early-MSD vs. late-MSD, p = 0.0031). No significant effects on the number of rearing behaviors nor the time spent in the center area were detected  = 2.314, p = 0.1050). One day after the training, recent fear memory was analyzed by contextual and cued tests  = 3.196, p = 0.0458; post hoc Tukey’s test: control vs. early-MSD, p = 0.0357). To assess if this contextual fear memory persisted for a long period, we investigated contextual remote fear memory, which was examined 21 days after the conditioning. We found a non-significant but similar decrease in the mean freezing time in early-MSD mice  = 2.229, p = 0.1138). The cued test consisted of two phases: an altered phase in a novel chamber but without the auditory cue, and a cued phase with the cue. The test was performed 1 h after the contextual test for both recent and remote memory analyses. The late-MSD mice exhibited a significant decrease in the mean freezing time not only for the recent but also for the remote memory compared to control mice . The early-MSD mice showed a non-significant but similar decrease in the mean freezing time for recent and remote memory. After the remote memory test, mice were exposed to the same conditions as the training sessions but without electrical shocks and were tested for contextual and cued memory. After the extinction of fear memory, the mean freezing times became lower in all groups with no significant differences between them  session, the mice exhibited reliable fear conditioning as measured by an increase in freezing time throughout the conditioning . Freezined tests . In the een them  right.F = 1.545, p = 0.2330). In the probe test, we could not detect any significant differences regarding the time spent around the target hole  = 0.5784, p = 0.5681) nor accuracy of the reference memory  = 0.5953, p = 0.5681) among the groups  = 0.5451, p = 0.5865) nor in the time spent around the target hole  = 0.1619, p = 0.8514) nor accuracy of the reference memory  = 2.140, p = 0.1387) in the probe test (The formation and maintenance of contextual and cued fear memory are largely mediated by the function of the hippocampus and amygdala . To disce groups . In addiobe test . These rIn the present study, we show for the first time that early life stress exhibits distinct effects on the activity of the NSC-neurogenesis system in the adult brain, depending on the timing and duration of the stress. MSD during the SHRP increases the size of the NSC population compared to controls; however, the same stress extended beyond the SHRP has the opposite effects. We presented two lines of evidence for the expansion of the NSC population after early-MSD: the estimated number of total NSCs using the neurosphere assay and the BrdU label retention assay in the SEZ. In the DG of the hippocampus, the estimation of NSCs pool was analyzed by BrdU long-term labeling co-expressed with GFAP and negative for S100β in the SGZ at 8 weeks. There have been very few studies to date evaluating NSC population size after early life events; however, our results are consistent with data obtained in another rodent study . During We also demonstrated an increase in neurogenesis in the BLA in addition to the dentate gyrus of the hippocampus, a classical neurogenic region, after early-MSD. Although amygdalar development is mostly complete by E15.5 in mice , neurogeN-methyl-D-aspartate type glutamate receptor (Currently, enhanced adult neurogenesis is thought to facilitate the clearance of fear memory and improve coping and recovery from psychological damage caused by stress, which could explain the resilience to stress . Repeatereceptor . Similarreceptor . Thus, tThis article is dedicated to Dr. Kazuhiro Ikenaka who passed away during the preparation of this manuscript.All datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by the Animal Care and Use Committee of the Shiga University of Medical Science.NK and SH designed the research. KD, TF, NK, NM, and SH performed the research. KD, NK, and SH analyzed the data and prepared the manuscript. KI and SH supervised the research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Enterovirus A71 (EV-A71) infection is primarily responsible for fatal hand, foot, and mouth disease (HFMD) cases. Infants and younger children are more likely to suffer central nervous system damage as a result of EV-A71 infection, but this virus mostly does not affect older children and adults. This study investigated the possible mechanism underlying the age-dependent lethal effect of EV-A71 infection by comparing neonatal and adult mouse models of EV-A71 infection. Although viral proliferation is absent in both neonatal and adult mice, we observed that EV-A71, as a stimulus for astrocytes, elevates the levels of cytokines and monoamine neurotransmitters in neonatal mice. Then, we selected IL-6 and adrenaline as targets in a pharmacological approach to further validate the roles of these factors in mediating the mortality of neonatal mice after EV-A71 infection. Intracerebral injection of IL-6 and adrenaline enhanced the severity of EV-A71 infection, while treatment with an anti-IL-6-neutralizing antibody or the adrenergic-antagonist phenoxybenzamine reversed the lethal effect of EV-A71 in neonatal mice. These results suggest that the central nervous system (CNS) damage in neonatal cases of EV-A71 infection might be caused by an activated fetal cerebral immune response to the virus, including the disruption of brainstem function through increased levels of cytokines and neurotransmitters, rather than the typical cytopathic effect (CPE) of viral infection. The central nervous system damage in neonatal mice with EV-A71 infection may be caused by activated fetal cerebral astrocytes related to the immune response to the virus, including the disruption of the functioning of the brainstem through increased cytokines and neurotransmitters, rather than the typical cytopathic effect of viral infection. Since 1997, hand, foot, and mouth disease (HFMD) has been an infectious disease that seriously affects the health of children in East and Southeast Asia . In seveIn our previous work, by using a rhesus macaque infection model and human autopsy, we found that EV-A71 can enter the CNS and generate a severe inflammatory response . AstrocyHerein, we first duplicated the dynamic progression of EV-A71 infection in the CNS of neonatal pups and adult mice. In addition, histopathological examinations were performed to determine whether astrocytes are involved in EV-A71 infection in neonatal mice. Then, we cultured murine astrocytes and described the viral growth curve and biological characteristics in infected cell cultures. In vivo investigations with the mouse model were performed to confirm that EV-A71-infected astrocytes were associated with the increased expression of proinflammatory cytokines and adrenaline in the brain. In addition, we chose IL-6 and adrenaline as targets in a pharmacological approach to further validate the roles of these factors in mediating the mortality of neonatal mice subjected to EV-A71 infection. This study collected data through in vitro and in vivo systems and compared neonatal mice with adults. If EV-A71 infection induces astrocyte dysfunction and the expression of cytokines and monoamine neurotransmitters is associated with its lethal effect, then this situation could be reversed by anti-IL-6-neutralizing antibodies and adrenergic antagonists.2. The culture medium was changed to DMEM supplemented with 2% FBS after viral infection. The EV-A71 strains were also titered on Vero cells.The EV-A71 virus strains FY-22 (GenBank: EU913466) and FY-23 (GenBank: EU812515) were used in this study. These strains belong to subgenogroup C4. FY-22 and FY-23 were isolated from the respiratory tract secretions of a patient with a mild EV-A71 infection and a child with severe cardiopulmonary collapse, respectively, in Fuyang, China, in May 2008 [2. The presence of a CPE was recorded at 7 days post infection (dpi).Virus titration was performed using a microtitration assay according to a standard protocol . BrieflySpecific-pathogen-free imprinting control region mice  were obtained from the Department of Small Animal of the Institute of Medical Biology, Chinese Academy of Medicine Sciences. The mice were singly housed in polycarbonate cages under 12 h light/dark conditions (lights on between 8:00 a.m. and 8:00 p.m.). Neonatal mice remained with the dams during the experimental phase. The room temperature was maintained at 22 ± 2 °C, and food and water were available ad libitum. All experimental procedures were reviewed and approved by the Yunnan Provincial Experimental Animal Management Association  2011–0005) and the Experimental Animal Ethics Committee of the institute .2. The primary cell culture was grown to confluence and washed three times with 0.01 M PBS before the addition of 0.0625% trypsin. The digested cells were centrifuged and suspended for fibroblast removal by differential adhesion for 30 min. The suspension was transferred to another culture plate for adhesion for 60 min, followed by the removal of microglia and oligodendroglia to obtain purified astrocytes, which were identified with green fluorescent anti-glial fibrillary acidic protein (GFAP) antibody following three rounds of purification.Astrocytes were separated from the cortex, mid brain, pons, and medulla oblongata of mice and cultured as previously described . BrieflyAstrocytes were infected with EV-A71 (multiplicity of infection = 0.1) for 60 min at 37 °C and washed twice with PBS. Then, 2% FBS was added prior to continuous incubation. The cell culture supernatants were harvested at different time points and stored at −80 °C prior to further analyses.RPL13A  were 5′-CCTTGGAGGAGAAGAGGAAAGAGA-3′ and 5′-TTGAGGACCTCTGTGTATTTGTCAA-3′.Viral RNA was extracted from EV-A71-infected astrocytes or Vero cells using the MiniBEST Viral RNA/DNA Extraction Kit  according to the manufacturer’s instructions. The presence of EV-A71 negative-strand RNA was detected using a PrimeScript RT reagent Kit  on a Mastercycler nexus GSX1 thermal cycler , and qRT-PCR amplification was performed using Taqman 1-step RT-PCR Master Mix  on a 7500 Fast Real-time RT-PCR system , as previously described , 16. TheThe cytokine levels in the culture supernatants and homogenized brain samples were evaluated using ELISA kits . Adrenaline levels were analyzed with a 3-CAT Research EIA Kit . These tests were carried out according to the manufacturer’s instructions. A sample volume of 100 μl was used to determine the cytokine levels in the culture supernatant. For the determination of cytokines in brain homogenates, the brain tissue was weighed, and an appropriate amount of lysis buffer was added to achieve 100 mg tissue/75 μl buffer; then, the sample was ground, homogenized, and centrifuged, and the supernatants were transferred. Next, 100 μl of 20–50-fold diluted supernatants were added to ELISA plates. For the determination of adrenaline in the brain homogenates, 800 μl PBS (with 1 mM EDTA) was added to a weighed sample of brain tissue; the sample was ground, homogenized, and centrifuged. The supernatants were then transferred, and the sample size was 200 μl for the subsequent assay.All neonatal mice (within 48 h of birth) were intracranially injected at the midpoint between the outer edge of the eye and the leading edge of the external ear, and adult mice (6 weeks old) were intracranially injected at an oblique orientation above the canthus . The inj4.5 TCID50/animal) and subsequently sacrificed at different time points post injection. In the infection experiment, neonatal mice were randomly divided into EV-A71-infected, inactivated virus, and control groups. The mice in the infected group were injected with EV-A71 , the mice in the inactivated virus group were injected with inactivated virus , and the mice in the control group were injected with the same volume of PBS. The mice were sacrificed at 4 dpi, and the brains were removed for subsequent pathological examination.To detect the virus loads in the mouse brain, neonatal or adult mice were intracerebrally injected with EV-A71 (104 CCID50 per mouse), or PBS. The survival of the mice was recorded over 7 dpi (for the FY-23 groups) or 10 dpi (for the FY-22 groups).For the quantification of mouse survival, neonatal mice were randomly divided into different groups and treated with IL-6 , adrenaline , an anti-IL-6-neutralizing antibody , phenoxybenzamine , or PBS. Ten minutes later, all mice were infected with a strongly virulent strain (FY-23), a weakly virulent strain (FY-22) of EV-A71  and a secondary horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody  in immunohistochemical analyses or an Alexa Fluor 594-conjugated donkey anti-mouse IgG antibody  in immunofluorescence assays. Astrocytes were detected using a rabbit anti-GFAP antibody  as the primary antibody and an Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody  as the secondary antibody. The staining procedure was performed according to a standard protocol , 18. Thet-tests. The mouse survival data were quantified and analyzed by the log-rank test. For all comparisons, a significant difference was assumed at p ≤ 0.05; all of the data were analyzed using SPSS version 21 or GraphPad Prism 7.00.All data except survival rates are expressed as the mean + SD (or SEM), and the differences between the two groups were evaluated using independent samples In our pilot work and previous studies , 19, theps < 0.05) within 64 hpi in astrocytes from mice , were found in the brain homogenates of neonatal mice treated with live EV-A71   Fig. . In comp05) Fig.  did not p < 0.05, log-rank test)  that causes death in some mice (37.50–43.75%) within 10 dpi Fig. , while ast) Fig. . TogetheThis study reveals the potential mechanisms responsible for the lethal effect of EV-A71 intracerebral injection in neonatal mice. As we report in the present study, neonatal mice died within 7 dpi, but there is no viral proliferation. This lethal effect of EV-A71 injection in neonatal mice is partly due to astrocyte dysfunction, following the increased expression of cytokines, the stimulation of neurons by these upregulated cytokines may lead to a disturbed homeostasis of monoamine neurotransmitters . AstrocyIn our previous study , to inveA fundamental issue related to EV-A71 infection of the CNS is the naturally targeted cells in the CNS. Considering the effects of poliovirus , which cWe observed that EV-A71, as an astrocyte stimulus, elevates the level of cytokines; then, the increased cytokines stimulate neuronal secretion of the monoamine neurotransmitter adrenaline . The disSupplemental Figure